Nanometer composition loaded with circA2ML1 and paclitaxel as well as preparation method and application of nanometer composition
By preparing nanocomposites loaded with circA2ML1 and paclitaxel, the problems of circRNA delivery stability and paclitaxel monotherapy resistance were solved, achieving targeted therapy and synergistic anti-tumor effects for ESCC and reducing toxic side effects.
Patent Information
- Application Number
- CN202511952166.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-02-17
AI Technical Summary
In existing technologies, circRNA is easily degraded by RNase in serum, and traditional delivery methods cannot target tumor tissue. Paclitaxel alone cannot achieve the synergistic effect of gene therapy and chemotherapy, and is prone to drug resistance and toxic side effects.
Nanocomposites loaded with circA2ML1 and paclitaxel were prepared and assembled with paclitaxel liposomes via polyurethane/circA2ML1 nanocomposites to form nanocomposites with a diameter of 130-150 nm for targeted therapy of esophageal squamous cell carcinoma.
It significantly inhibits ESCC tumor growth, reduces toxic side effects, achieves synergistic effects of circA2ML1 and paclitaxel, improves treatment efficacy, and has good biocompatibility.
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Abstract
Description
Technical Field
[0001] This invention relates to a nanocomposite loaded with circA2ML1 and paclitaxel, its preparation method and application, belonging to the field of biomedical technology. Background Technology
[0002] Esophageal cancer is a common malignant tumor worldwide, with esophageal squamous cell carcinoma (ESCC) accounting for the majority. Its incidence and mortality rates remain high, posing a serious threat to public health. Current single-drug chemotherapy treatments are prone to drug resistance due to compensatory activation. Therefore, further investigation into the key driver genes in the occurrence and development of ESCC is of great significance for its diagnosis and targeted therapy.
[0003] Circular RNA (circRNA) is a newly discovered non-coding RNA, a covalently closed transcript formed by backsplicing of precursor mRNA. circRNAs are characterized by high stability, high abundance, and high conservation, and have been reported to participate in various cancer-related biological processes. Based on these characteristics, circRNAs are emerging as potential diagnostic, prognostic, and therapeutic targets. However, circRNAs are easily degraded by RNase in serum, have a half-life of <2 hours, and exhibit clearance rates >80% within 1 hour after intravenous injection using traditional delivery methods (such as naked RNA), and cannot target tumor tissue.
[0004] Existing nanoscale circRNA delivery systems mainly include: ① single liposomes encapsulating chemotherapeutic drugs (such as PLD, doxorubicin liposomes), which improve efficacy by prolonging circulation time, but lack synergistic effects with gene therapy; ② polymer nanoparticles encapsulating siRNA for gene silencing, but the transient effect of siRNA makes it difficult to achieve sustained efficacy. For example, when using PEI to encapsulate siRNA, the high charge density leads to cytotoxicity, and it cannot simultaneously deliver genes and chemotherapeutic drugs. In addition, M2 macrophage polarization in the ESCC tumor microenvironment depends on AKT1 signaling, and traditional therapies cannot simultaneously regulate gene expression and the immune microenvironment.
[0005] Paclitaxel (PTX) is a tetracyclic diterpenoid secondary metabolite, primarily derived from plants of the Taxus genus. It is currently recognized as one of the most effective anti-tumor chemotherapy drugs in the world, effectively treating various cancers such as breast cancer, pancreatic cancer, and head and neck squamous cell carcinoma. However, as a first-line chemotherapy drug, paclitaxel (PTX) alone cannot achieve the synergistic effect of gene therapy and chemotherapy. Monotherapy easily leads to drug resistance in tumor cells; for example, the resistance rate of ESCC cells to paclitaxel can reach 35-40%. Furthermore, paclitaxel monotherapy requires high doses to ensure efficacy; the commonly used clinical dose is 135-175 mg / m². 2It can cause grade 3-4 neutropenia (occurring in about 60%) and peripheral neuropathy (about 30%), limiting the treatment duration and dosage.
[0006] Therefore, there is an urgent need to develop a novel nanocomposite that can solve problems such as insufficient synergistic therapy, low delivery efficiency, and toxic side effects. Summary of the Invention
[0007] To address the shortcomings of existing technologies, this invention provides a nanocomposite loaded with circA2ML1 and paclitaxel, its preparation method, and its application.
[0008] The technical solution of the present invention is as follows:
[0009] A nanocomposite loaded with circA2ML1 and paclitaxel is assembled from polyurethane / circA2ML1 nanocomposites and paclitaxel liposomes;
[0010] The nucleotide sequence of circA2ML1 is shown in SEQ ID NO.1.
[0011] According to a preferred embodiment of the present invention, the polyurethane / circA2ML1 nanocomposite is prepared according to the following method:
[0012] The circA2ML1 solution and the polyurethane solution were vortexed for 10-20 min, and then allowed to stand at 4℃ for 25-35 min to obtain the polyurethane / circA2ML1 nanocomposite.
[0013] More preferably, the concentration of the circA2ML1 solution is 0.8~1.2 μg / μL, the concentration of the polyurethane solution is 4.5~5.5 μg / μL, and the mass ratio of the polyurethane solution to the circA2ML1 solution is 1:(1~1.1).
[0014] Most preferably, the concentration of the circA2ML1 solution is 1 μg / μL, the concentration of the polyurethane solution is 5 μg / μL, and the mass ratio of the polyurethane solution to the circA2ML1 solution is 1:1.
[0015] More preferably, the polyamino ester is a poly(β-amino ester).
[0016] According to a preferred embodiment of the present invention, the paclitaxel liposome is an existing paclitaxel liposome used clinically.
[0017] The preparation method of the above-mentioned nanocomposite loaded with circA2ML1 and paclitaxel includes the following steps:
[0018] A polyurethane / circA2ML1 nanocomposite solution was added to a paclitaxel liposome solution to obtain a reaction solution. The resulting reaction solution was magnetically stirred at 35-45℃ and 550-650rpm for 1.5-2h to obtain a nanocomposite loaded with circA2ML1 and paclitaxel.
[0019] According to a preferred embodiment of the present invention, the concentration of circA2ML1 in the reaction solution is 1.2~1.8 nmol / μL, and the concentration of paclitaxel is 4.5~5.5 nmol / μL.
[0020] More preferably, the concentration of circA2ML1 in the reaction solution is 1.5 nmol / μL, and the concentration of paclitaxel is 5 nmol / μL.
[0021] The diameter of the nanocomposite prepared by the present invention, which is loaded with circA2ML1 and paclitaxel, is 130~150 nm.
[0022] The above-mentioned nanocomposition loaded with circA2ML1 and paclitaxel is used in the preparation of drugs for targeted treatment of esophageal squamous cell carcinoma.
[0023] A targeted therapy for esophageal squamous cell carcinoma, the drug comprising a nanocomposition loaded with circA2ML1 and paclitaxel.
[0024] Beneficial effects:
[0025] 1. The nanocomposite of circA2ML1 and paclitaxel provided by this invention has good anti-tumor function and can significantly inhibit the growth of ESCC tumors. Furthermore, circA2ML1 and paclitaxel have a synergistic effect, and the inhibitory effect on ESCC tumors is significantly enhanced when the two are used in combination compared with the use of circA2ML1 or paclitaxel alone.
[0026] 2. The nanocomposite loaded with circA2ML1 and paclitaxel provided by this invention exhibits significantly reduced toxic side effects and good in vivo biocompatibility. Animal experiments of this invention show that no pathological damage was observed in major organs after using the nanocomposite loaded with circA2ML1 and paclitaxel, confirming its good biocompatibility. Attached Figure Description
[0027] Figure 1 The analytical results are for the nanocomposite of nanomaterials loaded with circA2ML1 and paclitaxel prepared in Example 2;
[0028] In the figure, A represents the observation results of transmission electron microscopy (TEM), with a scale bar of 200 nm; B represents the results of dynamic light scattering analysis.
[0029] Figure 2 The results of tumor inhibition in mice treated with different drugs were shown for nanocomposites loaded with circA2ML1 and paclitaxel.
[0030] In the figure, A shows representative images of subcutaneous xenograft tumors in mice treated with different drugs; B shows volume analysis of subcutaneous xenograft tumors in mice treated with different drugs; and C shows weight analysis of subcutaneous xenograft tumors in mice treated with different drugs.
[0031] Figure 3 The tumor proliferation index of mice in different drug treatment groups.
[0032] Figure 4 Biosafety evaluation of nanocomposites loaded with circA2ML1 and paclitaxel;
[0033] In the figure, A shows the HE staining results of the lungs, liver, spleen, and kidneys of mice in different drug treatment groups (scale bar = 100 μm); B shows the quantitative analysis of blood cell counts in mice in different drug treatment groups; the error bar represents the standard deviation; ns indicates no statistically significant difference. Detailed Implementation
[0034] The technical solution of the present invention will be further described below with reference to experimental examples, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and materials involved in the examples are all commercially available products.
[0035] The human esophageal cancer cell line KYSE-410 is available from Fuheng Biotechnology Co., Ltd.
[0036] The polyamino ester used in the examples is poly(β-amino ester), abbreviated as PβAE, which is available from Xi'an Ruixi Biotechnology Co., Ltd.
[0037] The paclitaxel liposomes used in the examples are existing paclitaxel liposomes used clinically and are available from Qilu Pharmaceutical Co., Ltd.
[0038] Example 1
[0039] 1. Beijing Qingke Biotechnology Co., Ltd. was commissioned to artificially synthesize the nucleotide sequence shown in SEQ ID NO.1. Specifically, the nucleotide sequence was to form a circular RNA with the first and second ends joined together. The linear RNA was removed by enzymatic digestion. Finally, the circA2ML1 solution with a concentration of 1 μg / μL and A260 / A280=2.0 was purified.
[0040] 2. Dissolve 5 mg of poly(β-amino ester) in 1 mL of NaOAc buffer (50 mM, pH=4) to prepare a 5 μg / μL poly(β-amino ester) solution.
[0041] Example 2
[0042] A method for preparing a nanocomposite loaded with circA2ML1 and paclitaxel includes the following steps:
[0043] (1) The circA2ML1 solution (1 μg / μL) prepared in Example 1 and the poly(β-amino ester) solution (5 μg / μL) were vortexed at a mass ratio of 1:1 for 15 min, and then allowed to stand at 4 °C for 30 min to obtain the poly(β-amino ester) / circA2ML1 nanocomposite solution (PBAE / circA2ML1).
[0044] (2) Add paclitaxel liposomes to a rotary evaporator and remove the organic solvent completely by vacuum evaporation. Then add deionized water and hydrate at 40°C for 30 min to obtain paclitaxel liposome solution.
[0045] (3) The poly(β-amino ester) / circA2ML1 nanocomposite solution was added to the paclitaxel liposome solution to obtain a reaction solution; the resulting reaction solution was magnetically stirred at 40℃ and 600rpm for 2h to complete the assembly and obtain a nanocomposite (PLPC) loaded with circA2ML1 and paclitaxel.
[0046] The concentration of circA2ML1 in the reaction solution is 1.5 nmol / μL, and the concentration of paclitaxel is 5 nmol / μL.
[0047] The PLPC prepared in this example was observed by transmission electron microscopy, and the results are as follows: Figure 1 As shown in A. Then, dynamic light scattering analysis was performed on the PLPC prepared in this embodiment using a Malvern Zetasizer Nano ZS90 instrument to determine its particle size. The results are as follows: Figure 1 As shown in B.
[0048] Depend on Figure 1 As shown in A~B, the PLPC prepared in this embodiment has a diameter of 130~150nm, an average particle size of about 140nm, a PDI<0.2, and exhibits sufficient stability at room temperature.
[0049] Example 3
[0050] A method for preparing a nanocomposite loaded with circA2ML1 and paclitaxel, the specific steps are the same as in Example 1, except that in step (3), the concentration of circA2ML1 in the reaction solution is 20 nmol / μL and the concentration of paclitaxel is 100 nmol / μL by adjusting the amount of poly(β-amino ester) / circA2ML1 nanocomposite solution and paclitaxel liposome solution.
[0051] Example 4
[0052] 1. Establishment of ESCC mouse model: 20 four-week-old female BALB / c nude mice were injected with 3×10⁻⁶ spores under their right axilla. 6 KYSE-410 cells were cultured in mice until the tumor volume reached 50 mm. 3 At that time, the ESCC mouse model was obtained.
[0053] Grouping: The ESCC mouse model was divided into 4 groups of 5 mice each: group 1 (PBS), group 2 (PBAE / circA2ML1), group 3 (paclitaxel liposomes) and group 4 (PLPC).
[0054] Administration: The four ESCC mouse models were administered the drug via tail vein injection, once every 3 days for a total of 5 times;
[0055] Group 1 was given an equal volume of PBS buffer, denoted as PBS.
[0056] Group 2 was administered PBAE / circA2ML1 at a final concentration of 100 µg / kg, denoted as PC;
[0057] Group 3 was administered paclitaxel liposomes at a final concentration of 5 mg / kg, denoted as PL.
[0058] Group 4 was administered PLPC (prepared in Example 4) containing a final concentration of circA2ML1 of 100 µg / kg and a final concentration of paclitaxel of 5 mg / kg;
[0059] 2. Tumor growth curve plotting
[0060] The tumor length (L) and width (W) of the four groups of mice were measured every 3 days, and the volume V was calculated as V = (L × W) / (L × W) 2 ) / 2, the result is as follows Figure 2 As shown in A~B.
[0061] Depend on Figure 2 As shown in A~B, the average tumor volume of group 4 mice on day 15 was 126.5±23.4 mm³, which was significantly smaller than that of group 2 mice (287.3±35.6 mm³). 3 The average length of mice in group 3 was 215.7 ± 28.9 mm. 3 The average length of the mice in group 1 was 458.6 ± 42.1 mm. 3 The p-value was <0.01. This indicates that the nanocomposite prepared by this invention, loaded with circA2ML1 and paclitaxel, has good anti-tumor activity and can significantly inhibit the growth of ESCC tumors in mice after administration via tail vein injection. Furthermore, the combined use of circA2ML1 and paclitaxel significantly enhances the inhibitory effect on tumor growth compared to using circA2ML1 or paclitaxel alone.
[0062] 3. Tumor weight measurement
[0063] Four groups of mice were sacrificed, tumors were removed, and the tumors were weighed. The results are as follows: Figure 2 As shown in C.
[0064] Depend on Figure 2 As shown in C, the average tumor weight of mice in group 4 was 0.32±0.05g, which was significantly reduced by 53% and 37% compared to 0.68±0.08g in group 2 and 0.51±0.06g in group 3, respectively. This further demonstrates that the combined use of circA2ML1 and paclitaxel has a significant inhibitory effect on tumor growth.
[0065] 4. Immunohistochemistry
[0066] (1) Sample processing: Tumor tissues from 4 groups of mice were fixed in formaldehyde, embedded in paraffin, and serially sectioned with a thickness of 4 μm. The sections were then treated with xylene and graded ethanol to dewax them to water, resulting in dewaxed sections.
[0067] (2) Antigen retrieval: The dewaxed sections were placed in citrate buffer and antigen retrieval was performed by high temperature and high pressure or microwave to expose the antigen epitopes. After retrieval, the sections were cooled and washed to obtain antigen-retrieval sections.
[0068] (3) Blocking and incubation: Incubate the antigen-repaired sections with 3% hydrogen peroxide solution at room temperature to block endogenous peroxidase activity, add primary antibody (Ki67), incubate overnight at 4°C, wash, add the corresponding secondary antibody (horseradish peroxidase), and incubate at room temperature for 30~60 min;
[0069] (4) Staining and Counterstaining: Add DAB chromogenic agent to the sections obtained in step (3) and react until the brownish-yellow signal is clear. Stop the staining with distilled water. Counterstain the cell nuclei with hematoxylin. After differentiation with hydrochloric acid and ethanol, ammonia water turns the stain blue. Dehydrate with graded ethanol, clear with xylene, and mount with neutral resin. Observe the expression location (nucleus, cytoplasm, or cell membrane) and staining intensity of the target protein in the sections using an optical microscope. Perform semi-quantitative analysis after image acquisition (e.g., scoring based on the proportion of positive cells and staining depth). The results are as follows: Figure 3 As shown.
[0070] Depend on Figure 3 It was found that the Ki67 positivity rate in group 4 tumor tissue was 21.3%, significantly lower than that in other groups. This indicates that the nanocomposite prepared by this invention, loaded with circA2ML1 and paclitaxel, has good anti-tumor activity, and the combined use of circA2ML1 and paclitaxel has a significant inhibitory effect on tumor growth, which is significantly improved compared to the use of circA2ML1 or paclitaxel alone.
[0071] Example 6
[0072] After the mice in Example 5 were euthanized, major organs such as the lungs, spleen, kidneys, and liver were collected. These organs were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (approximately 4 μm thick), stained with hematoxylin and eosin (H&E), and observed and photographed under a microscope to observe histological morphological changes. Simultaneously, peripheral blood was collected from the mice, and the levels of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), neutrophils (NEU), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) were measured using an automated blood analyzer. The results are as follows: Figure 4 As shown.
[0073] Depend on Figure 4 It was found that the blood routine (white blood cell count, platelet count, etc.) of mice in group 4 were not significantly different from those in group 1. HE staining showed no obvious pathological damage to the liver, spleen, lungs, and kidneys. The results indicate that the composition of the present invention has no adverse effects on the structure of major organs and tissues and hematological indicators of mice under experimental administration conditions, and has good in vivo biocompatibility.
[0074] The embodiments described above are merely preferred implementations of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A nano-composition loaded with circA2ML1 and paclitaxel, characterized in that, is assembled by polyaminoester / circA2ML1 nanocomplex and paclitaxel liposome; The nucleotide sequence of the circA2ML1 is shown as SEQ ID NO.
1.
2. The nano-composition loaded with circA2MLl and paclitaxel according to claim 1, wherein, The polyaminoester / circA2ML1 nanocomplex is prepared according to the following method: The circA2ML1 solution and the polyaminoester solution are vortexed and mixed for 10-20 min, and then are left to stand at 4℃ for 25-35 min to obtain the polyaminoester / circA2ML1 nanocomplex.
3. The nano-composition loaded with circA2MLl and paclitaxel as claimed in claim 2, wherein, The concentration of the circA2ML1 solution is 0.8-1.2 μg / μL, and the concentration of the polyaminoester solution is 4.5-5.5 μg / μL; the mass ratio of the polyaminoester solution to the circA2ML1 solution is 1: (1-1.1).
4. The nano-composition loaded with circA2MLl and paclitaxel as claimed in claim 3, wherein, The concentration of the circA2ML1 solution is 1 μg / μL, and the concentration of the polyaminoester solution is 5 μg / μL; the mass ratio of the polyaminoester solution to the circA2ML1 solution is 1:
1.
5. The circA2MLl and paclitaxel loaded nanoparticle composition of claim 2, wherein, The polyaminoester is poly(β-amino ester).
6. A method of preparing the nano-composition of claim 1 loaded with circA2MLl and paclitaxel, characterized in that, The method comprises the following steps: The polyaminoester / circA2ML1 nanocomplex solution is added to the paclitaxel liposome solution to obtain a reaction liquid; the obtained reaction liquid is magnetically stirred at 35-45℃ and 550-650 rpm for 1.5-2 h to obtain a nanocomposition loaded with circA2ML1 and paclitaxel.
7. The production method according to claim 6, wherein The concentration of circA2ML1 in the reaction liquid is 1.2-1.8 nmol / μL, and the concentration of paclitaxel is 4.5-5.5 nmol / μL.
8. The production method according to claim 7, characterized in that, The concentration of circA2ML1 in the reaction liquid is 1.5 nmol / μL, and the concentration of paclitaxel is 5 nmol / μL.
9. The use of the nanocomposition loaded with circA2ML1 and paclitaxel according to claim 1 in the preparation of a drug for targeted treatment of esophageal squamous cell carcinoma.
10. A drug for targeted treatment of esophageal squamous cell carcinoma, characterized by, The drug comprises the nanocomposition loaded with circA2ML1 and paclitaxel.